The human body encounters a number of chemical exposures on a daily basis, which may have short- or long-term health implications. Previously it has been demonstrated that the entire respiratory tract of an individual reacts to exposures like tobacco smoke in a similar manner, and that common molecular changes can be measured in airway epithelium. I propose that cataloguing the exposure of airway epithelial cells to tobacco cigarette (TCIG) smoke and its constituents, electronic cigarette (ECIG) aerosol and other drugs and small molecules can significantly increase the understanding of chemical exposure and identify common gene expression alterations. First, I determined the molecular impact of ECIG aerosol exposure on human airway epithelium in vitro, including alterations in genes related to xenobiotic metabolism, oxidative stress, and ciliated cells. These changes were generally less pronounced than the effects of TCIG exposure, and were more pronounced in ECIG products containing nicotine than those without nicotine. Furthermore, gene expression differences observed in vitro were concordant with differences observed in airway epithelium collected from ECIG users. Second, I examined the impact of TCIG exposure and TCIG constituents on premalignant airway cells, to better understand the progression or regression of precancerous lesions. These data could also identify the constituents of TCIGs and the precancerous mutations that increase the risk for malignancy. Third, in an effort to build a high-throughput methodology for chemical exposures, I exposed primary lung cell lines to small molecule therapeutics and identified lung-specific and lung cell-type-specific effects of exposure, suggesting that profiling additional cell lines would further inform airway gene expression in response to exposure and that organ-specific exposure profiling may provide valuable insight into drug discovery for common diseases. Overall, transcriptomic profiles from the airway epithelium reflect exposure to various inhaled and chemical perturbations. These gene expression profiles indicate common changes across a multitude of airway exposures as well as unique alterations specific to a given perturbation. Gene expression profiling can therefore be used to detail the potential response to a compendium of chemical exposures including those that are either well-established or potential risk factors for chronic lung diseases. / 2019-07-09T00:00:00Z

yes / A major challenge in the production of 3D tissue engineered skin is the recreation of the basement membrane region to promote secure attachment and yet segregation of keratinocytes from the dermal substitute impregnated with fibroblasts. We have previously shown that simple electrospun scaffolds provide fibres on which the cells attach, proliferate, and self-sort into epithelium and dermis. In a development of this in this study tri-layered scaffolds were then electrospun from poly L-lactic acid and poly hydroxybutyrate-co-hydroxyvalerate. In these a central layer of the scaffolds comprising nano-porous/nano-fibrous poly hydroxybutyrate-co-hydroxyvalerate fibres was interwoven into the bulk micro-porous poly L-lactic acid microfibers to mimic the basement membrane. Keratinocytes and fibroblasts seeded onto these scaffolds and cultured for 2 weeks showed that neither cell type was able to cross the central nano-porous barrier (shown by SEM, and fluorescence monitoring with CellTracker™) while the micro-fibrous poly L-lactic acid provided a scaffold on which keratinocytes could create an epithelium and fibroblasts could create a dermal substitute depositing collagen. Although cells did not penetrate this barrier the interaction of cells was still evident-essential for epithelial development.

No / To investigate whether there are differences between members of the Burkholderia cepacia complex in their ability to invade human respiratory epithelial cells, 11 strains belonging to genomovars I-V were studied in an antibiotic protection assay using the A549 cell line. Strains belonging to genomovars II and III were more invasive than those of genomovars I, IV and V. There was also intra-genomovar variation in invasiveness. No correlation between invasiveness and other putative virulence factors of importance in B. cepacia infection in individuals with cystic fibrosis, cable pilus and B. cepacia epidemic strain marker was identified.

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Epithelial tissues are one of the most abundant tissues in our body. They make up essential organs like the gut, heart and eyes. These organs take up their complex 3D shapes during normal development of the embryo. Our understanding of such large-scale 3D shape changes is limited mainly due to the technical difficulties of imaging and quantifying such developmental events. In this thesis, I study two events in which epithelial monolayers change their 3D shape. In both the projects, I use data from light-sheet microscopic images of developmental events. These data are provided by my collaborators. In this thesis, I further analyzed them using quantitative approaches and interpreted them using computational models. In the first project, I study a case of a developing tissue inside a rigid confinement. A perfect model system for this is the Drosophila embryo which consists of an epithelial monolayer (blastoderm) inside a rigid shell (vitelline membrane). During gastrulation, the blastoderm is under compressional stresses due to tissue proliferation and compression from the germband extension. During this time, an invagination separating the future head and the trunk region appears. This is known as the cephalic furrow (CF). As the CF disappears after some time, its relevance in the normal development of the embryo is unclear. To understand its role, my collaborators image the blastoderm in mutant embryos which lack CF. These mutant embryos have either of the genes even-skipped (eve) or buttonhead (btd) knocked down. In the absence of CF, temporary ectopic folds appear in the blastoderm in locations which vary between embryos. Unlike the CF, ectopic folds appear suddenly and hence look like buckling events. I hypothesize that ectopic folds appear because of the compressive stresses generated in the blastoderm during the germband extension or by the compression of tissues that are adjacent to mitotic domains. Moreover, in normal embryos, CF, which is a controlled invagination, acts as a sink for the compressive stresses and thus suppresses ectopic folds. To test this hypothesis, I modelled the blastoderm as a 2D elastic tissue which is confined inside a rigid boundary acting as the vitelline membrane. In my model, I show that the stresses generated by both the germband extension and the mitotic domains contribute to the formation of ectopic folds. I model the CF as a region with some preferred intrinsic curvature, thus acting as a programmed fold. I show that ectopic folds are inhibited in the presence of a CF. However, the efficiency of the CF depends on the strength of the CF and, interestingly, the timing of the CF. I observe that even a weak CF can inhibit ectopic folds if it appears before the appearance of mitotic domains. I speculate that this could explain why the CF appears before the mitotic domains in the Drosophila embryo. n the second project, I study a case of shape change associated with the development of the Drosophila wing. Here I focus on the wing disc pouch, an epithelial monolayer that forms the adult wing blade. During metamorphosis, the larval wing disc evaginates to form the pupal wing. This process is known as eversion. During late larval stage, the wing disc pouch looks like a spherical cap. I refer to this stage as wL3 (wandering larval stage 3) in this thesis. Four hours after pupariation (4hAPF), the spherical cap deforms to an asymmetric dome such that it has a higher curvature along one cross-section compared to its perpendicular cross-section. Using segmented outlines of the wing in the two cross-sections of multiple images at different developmental stages, I compute the mean shape and quantify the curvature along the arclength of these shapes. To model this shape change, I use a 3D spring lattice whose initial curvature is matched to the curvature of the wL3 stage. Next, using apical cell shape data, provided by my collaborator, I compute a quantity referred to as a “spontaneous deformation tensor”. This tensor quantifies the amount of deformation, at a specific location between two developmental stages, due to cell area changes, cell elongation changes, and neighbour exchanges. I input this deformation pattern in my model which then changes its 3D shape. I find that the deformation due to changes in cell area and elongation increase the size of the tissue globally without affecting its curvature. However, the deformation due to cell rearrangements enhances curvature along one cross-section more than its perpendicular cross-section. Overall, the quantifications and modelling shows how different cellular behaviours deform the tissue locally. Moreover, a spatial pattern of different cellular behaviours can explain essential aspects of the shape change observed during the development of the wing.

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